This research program applies state-of-the-art mild chemical methods for peptide synthesis to prepare analogues of bovine pancreatic trypsin inhibitor (BPTI), which are then studied by a series of biophysical techniques in order to learn about structure, dynamics, and folding. BPTI is a small 58-residue protein with an array of three disulfide bridges. Extensive preliminary results have demonstrated the syntheses and characterizations of BPTI with the natural sequence, and of two analogues which replace paired half-cystine residues by paired alpha-amino-n-butyric acid (Abu) isosteres. It has been shown that whereas BPTI with all six half-cystines replaced by Abu (all-Abu-BPTI) behaves as a true random coil, BPTI with one intact disulfide between half-cystines 14 and 38 ([Cys14,38]-BPTI) is partially folded, with native-like structure in the hydrophobic core and disordered structure in the rest of the molecule. [Cys14,38]-BPTI represents a unique example of a protein which forms both a "molten globule" and an "A state;" these species have structures very similar to the transient intermediate formed in the first 10 to 20 msec of the protein folding process. Specific aims of this research are two-fold, seeking to synthesize and characterize proteins that are partially folded, and conversely, to synthesize and characterize circularized or circular permuted variants that may have enhanced stability. Proposed BPTI analogues may contain in a single molecule those sequences that encode initiation of folding and the final tertiary fold, for example by circularizing all-Abu-BPTI with a peptide bond connecting the N- and C- termini [these are proximal in native BPTI], by using oligoglycyl (or related) spacers to connect BPTI sequences corresponding to the slow- exchange core, or by substituting key residues in the core of [Cys14,38]- BPTI. The apparent "circularity" of BPTI, and the role of disulfide bridges in governing the protein's stability and postulated folding pathways, will be tested by syntheses of parent molecules and analogues of circularly permuted and circular BPTI' s with one or more disulfide bridges replaced by Abu residues, and/or side-chain lactam, thioether (carba), or dicarba bridges. Questions to be asked include: How does the nature of amino acids in the hydrophobic core affect its formation? How does the order of the secondary structural elements affect the stabilities, structures, and activities of the proteins? How does the order of the cysteine residues affect the acquisitions and stabilities of final conformations? What are the minimal covalent constraints required to achieve native-like conformations and activities? Can additional covalent constraints lead to more stable proteins? These studies represent a new approach to BPTI folding, and are expected to lead to significant insights that contribute to the "protein folding problem." A further goal of this work is to develop a general methodology for stabilizing therapeutically promising small proteins against denaturation and proteolysis, and for reducing their immunogenicity. Progress from this grant could impact on the development of drugs for the treatment of emphysema and arthritis, among other diseases.